(A) Example step response illustrating increasing duration analysis windows (above trace) used to compute the cumulative spike count for that one response (bottom). (B) Coincident ACh increases neuronal excitability in response to different step amplitudes. Plot of the cumulative spike count across different durations of analysis windows for weak (left panel) and moderate (right panel) step amplitudes. Individual points represent mean ± SEM over eight experiments with typically three trials per experiment in control (black) and ACh release (orange, light pulse train initiated 2 s before step). Example spike discharges evoked by the same depolarizing step shown above plot with vertical lines indicating the timing of each AP relative to the step. The total number of APs evoked by the step (right-most points in summary plot) differed significantly in response to both weak and moderate step intensities; *** 300 pA steps (left); p=6.04E-5, T = 8.530; 500 pA steps (right): p=7.31E-6, T = 11.754; both paired t-test. Inset: The increase in number of spikes between step and step + light vs. firing frequency during step only. *p=0.0130, T = 3.304, paired t-test. Orange arrow near X axis indicates time within depolarizing step responses when ACh increased the average cumulative spike count by 1 AP. The same analysis format was used in B, D, and in subsequent figures. (C) Diagram illustrating two potential mechanisms to explain long delay before ACh enhances neuronal excitability. The critical signaling event triggered by muscarinic receptors could be slow (red text), resulting in the ~ 700–900 ms delay, or the delay could reflect slow intrinsic kinetics of the ionic channels modulated by ACh (blue text). (D) ACh-modulated increase in neuronal excitability does not reflect muscarinic receptor signaling time. Plot of cumulative spike count in control conditions and with coincident ACh release (as in B) in seven experiments in which the light pulse train began 1 s (left panel), 2 s (middle) or 3 s (right) before the depolarizing step. **p<0.01 in all three panels, paired t-test; 1 s delay: T = 4.60.5973, 2 s: T = 5.199, 3 s: T = 6.579. E-G, Summary of the seven experiments illustrated in D. (E) The increase in number of spikes from the step to step + light conditions (illustrated as number of spikes) was not significantly different between different delays (n.s. p>0.05, 1 s vs. 2 s: p=1, T = 0.235, 1 s vs. 3 s: p=1, T = 0.328, 2 s vs. 3 s: p=1, T = 0. Paired t-test, Bonferroni corrected). (F) Plot represents mean bifurcation time (time when the step and step + light cumulative spike count plots diverged by at least 1 AP) calculated separately in each experiment and then averaged. (Bifurcation times presented in B and D represent times when the ensemble average plots diverged by at least 1 AP. Both bifurcation time protocols typically yielded similar latency estimates.) (G) Mean ISI ratio that assesses spike frequency adaptation across conditions. ISI ratio was calculated by the ratio between the average of the last 2 ISIs and the average of the first 2 ISIs. ISIs were significantly reduced by light induced ACh release (step vs. step + light: ** 1 s: p=0.00140, T = 7.808; ** 2 s: p=0.00206, T = 7.277; *p=0.0167, T = 4.874; paired t-test, Bonferroni corrected). Within step + light conditions, all comparisons between delays were not significantly different (1 s vs. 2 s: p=1, T = 0.520; 1 s vs. 3 s: p=0.936, T = 1.620; 2 s vs. 3 s: p=0.798, T = 1.735; paired t-test, Bonferroni corrected).